CFTR (cystic fibrosis transmembrane conductance regulator), encoded by the gene mutated in CF patients, is one of approximately 50 human ATP-binding cassette (ABC) proteins, and belongs to subfamily ABC-C which also includes SUR (sulfonylurea receptor) and MRP (multidrug resistance related) proteins. Unlike other ABC proteins, CFTR is an ion channel; it allows the Cl- flow needed for transepithelial fluid movement. Opening and closing of the CFTR channel pore are controlled by ATP binding to CFTR's two nucleotide binding domains (NBDs) and by ATP hydrolysis. Transduction of these NBD events to the channel gates is regulated by phosphorylation, by cAMP-dependent protein kinase, of multiple serines in CFTR's regulatory (R) domain. The goal of the proposed research is to understand, in molecular detail, the mechanisms regulating NBD function and channel gating. Knowing the precise mechanisms that control CFTR channel opening and closing might help pharmacological rescue in CF patients of cells with inadequate ion flow due to expression of mutant CFTR channels; this includes mutants that reach the cell surface in inadequate numbers, those with diminished pore conductance, and those that spend an insufficient time open. The specific aims are essentially unchanged. The first addresses what the NBDs look like, how they function, how they interact, and how they control the channel's gates. The working hypothesis is that CFTR's two NBDs are structurally dissimilar (a characteristic of ABC-C family members), that upon ATP binding they form head-to-tail dimers that enclose two ATP molecules in composite catalytic sites within the dimer interface, that the dimerization drives channel opening, and that hydrolysis of the ATP at the NBD2 catalytic site prompts channel closing; ATP remains bound at the NBD1 catalytic site for several minutes without being hydrolyzed. The second aim addresses how phosphorylation (and at which site or sites) permits channel opening, and how additional phosphorylation promotes stabilization of the channel open state. Wild-type and mutant CFTR channels will be expressed in oocytes and mammalian cells, and their structure and function analyzed using biophysical, electrophysiological, and biochemical methods. Mutant cycle measurements of single-channel gating kinetics will probe energetic interactions between residues and domains of CFTR. Photolabeling will probe nucleotide interactions with the NBDs. Structural analysis of prokaryotic NBD heterodimers, with an active and a dead catalytic site as in CFTR, will elucidate mechanisms in CFTR's NBDs.